Studies on an anti-trypanosomal factor (ATF) from the bacterial species, Pseudomonas fluorescences disclosed that countercurrent chromatography (CCC) employing the I to multilayer coiled column was very effective for the fractionation of larger samples of the crude product without decreasing resolution or prolonging the speed of separation. Significant lytic activity was produced by amounts of the purified fractions as low as 80 mug. It was also possible to separate two types of components, one that lysed the parasites and one that paralysed and killed, but did not lyse. The difference between these factors was established further on the basis of protein assays and ultrastructural studies of trypomastigotes of Trypanosoma equiperdum treated with these fractions, and with high performance liquid chromatography (HPLC). The CCC-HPLC system will aid in future research on the structure and synthesis of this antibiotic and its chemotherapeutic effect in experimental infections with T. cruzi.